Methods and Compositions for Increasing the Efficiency of Therapeutic Antibodies Using NK Cell Potentiating Compounds

ABSTRACT

The present invention relates, generally, to methods and compositions for increasing the efficiency of therapeutic antibodies. Their efficiency is enhanced through the increase of the ADCC mechanism. More particularly, the invention relates to the use of a therapeutic antibody in combination with compounds that block an inhibitory receptor or stimulate an activating receptor of an NK cell in order to enhance the efficiency of the treatment with therapeutic antibodies in human subjects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/897,624, filed Jul.23, 2004, now allowed, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/489,489, filed on Jul. 24, 2003, which is herebyincorporated by reference in their entirety, including all figures,tables, amino acid sequences and polynucleotide sequences.

FIELD OF THE INVENTION

The present invention relates, generally, to methods and compositionsfor increasing the efficiency of therapeutic antibodies. Moreparticularly, the invention relates to the use of a therapeutic antibodyin combination with a compound that blocks an inhibitory receptor orstimulates an activating receptor of natural killer cells, therebyallowing a potentiation of natural killer cell cytotoxicity in mammaliansubjects in order to enhance the efficiency of the treatment in humansubjects, particularly through an increase of the ADCC mechanism.

BACKGROUND OF THE INVENTION

Various therapeutic strategies in human beings are based on the use oftherapeutic antibodies. These include, for instance, the use oftherapeutic antibodies developed to deplete target cells, particularlydiseased cells such as virally-infected cells, tumor cells or otherpathogenic cells. Such antibodies are typically monoclonal antibodies,of IgG species, typically with human IgG1 or IgG3 Fc portions. Theseantibodies can be native or recombinant antibodies, and are often“humanized” mice antibodies (i.e., comprising functional domains fromvarious species, typically an Fe portion of human or non human primateorigin, and with a variable region or complementary determining region(CDR) of mouse origin). Alternatively, the monoclonal antibody can befully human through immunization in transgenic mice having the human Iglocus, or obtained through cDNA libraries derived from human cells.

A particular example of such therapeutic antibodies is rituximab(Mabthera®, Rituxan®), which is a chimeric anti-CD20 monoclonal antibodymade with human γl and κ constant regions (therefore with human IgG1 Feportion) linked to murine variable domains conferring CD20 specificity.In the past few years, rituximab has considerably modified thetherapeutical strategy against B lymphoproliferative malignancies,particularly non-Hodgkin's lymphomas (NHL). Other examples of humanizedIgG1 antibodies include alemtuzumab (Campath-1H®), which is used in thetreatment of B cell malignancies, and trastuzumab (Herceptin®), which isused in the treatment of breast cancer. Additional examples oftherapeutic antibodies under development are disclosed in the art.

The mechanism of action of therapeutic antibodies is still a matter ofdebate. Injection of antibodies leads to depletion of cells bearing theantigen specifically recognized by the antibody. This depletion can bemediated through at least three mechanisms: antibody mediated cellularcytotoxicity (ADCC), complement dependant lysis, and direct antitumorinhibition of tumor growth through signals given via the antigentargeted by the antibody.

While these antibodies represent a novel and efficient approach to humantherapy, particularly for the treatment of tumors, they do not alwaysexhibit a strong efficacy. For instance, while rituximab, alone or incombination with chemotherapy, was shown to be effective in thetreatment of both low-intermediate and high-grade NHL, 30% to 50% ofpatients with low grade NHL have no clinical response to rituximab. Ithas been suggested that the level of CD20 expression on lymphoma cells,the presence of high tumor burden at the time of treatment, or low serumrituximab concentrations may explain the lack of efficacy of rituximabin some patients. Nevertheless, the actual causes of treatment failureremain largely unknown.

Further, the use of therapeutic antibodies can be limited by sideeffects caused by their administration. For example, side effects suchas fever, headaches, nausea, hypotension, wheezing, rashes, infections,and numerous others can appear in patients, potentially limiting thepossible amount or frequency with which the antibodies can beadministered.

Thus, it would be very interesting to increase the efficacy oftherapeutic antibodies, or to be able to achieve therapeutic efficacyusing reduced doses of the antibodies that are less likely to produceside effects. The present invention addresses these and other needs.

SUMMARY OF THE INVENTION

The present invention discloses novel approaches to enhance the efficacyof therapeutic antibodies. Without being limited by the followingtheory, it is believed that the surprising results achieved using thepresent methods stem from their ability to enhance the ADCC mechanism invivo, when therapeutic antibodies are injected. Indeed, the presentinvention provides novel compositions and methods that overcome thecurrent difficulty related to the efficacy of therapeutic antibodies. Itis shown in the present invention that NK cells from an individual canhave poor therapeutic mAb (monoclonal antibody)-mediated ADCC because ofa lack of activation of NK cells, e.g., by an inhibition of inhibitoryreceptors on NK cells. Preferably, an increase of the ADCC mechanism isachieved by the administration of compounds that block an inhibitoryreceptor, or stimulate an activating receptor, on natural killer cells,thereby promoting a potentiation of natural killer cell cytotoxicity inmammalian subjects. Preferably the compound is an antibody or a fragmentthereof. Said antibodies or other compounds can react with an inhibitoryreceptor of NK cells, e.g., Killer inhibitory receptor (KIR or NKG2A/C)molecules, or with activating receptors, e.g., NCRs such as NKp30,NKp44, or NKp46, on NK cells, thereby neutralizing the inhibition of thecells and increasing their ADCC activity.

More specifically, the invention discloses methods of treatments of asubject in which a compound, preferably an antibody or a fragmentthereof, that blocks an inhibitory receptor or stimulates an activatingreceptor of an NK cell, is co-administered with the therapeutic antibodyto the subject. The inventors demonstrate here that the efficiency of atherapeutic antibody can be greatly enhanced by the co-administration,e.g., co-injection, of such a compound, preferably an antibody or afragment thereof, that overcomes the inhibition of NK cells, e.g., byblocking the inhibitory receptor or stimulating an activating receptorof an NK cell.

The invention also concerns pharmaceutical compositions comprising atherapeutic antibody and a compound, preferably an antibody or afragment thereof, that blocks an inhibitory receptor or stimulates anactivating receptor of an NK cell. The invention also concerns kitscomprising a therapeutic antibody and a compound, preferably an antibodyor a fragment thereof, that blocks an inhibitory receptor or stimulatesan activating receptor of an NK cell.

The invention also concerns the use of a compound, preferably anantibody or a fragment thereof that blocks the inhibitory receptor orstimulates an activating receptor of an NK cell, for increasing theefficiency of a treatment with a therapeutic antibody, or for increasingADCC in a subject submitted to a treatment with a therapeutic antibody.

The invention also concerns the use of a compound, preferably anantibody or a fragment thereof, that blocks the inhibitory receptor orstimulates an activating receptor of an NK cell, and of a therapeuticantibody for the preparation of a drug for treating a disease. Moreparticularly, the treatment of the disease requires the depletion of thetargeted cells, preferably the diseased cells such as virally-infectedcells, tumor cells or other pathogenic cells. Preferably, the disease isa cancer, infectious or immune disease. More preferably, the disease isselected from the group consisting of a cancer, an auto-immune disease,an inflammatory disease, and a viral disease. The disease also concernsa graft rejection, more particularly allograft rejection, and graftversus host disease (GVHD).

The present invention also comprises a method for reducing the dosage ofa therapeutic antibody, e.g., an antibody that is bound by an Feyreceptor, preferably CD16 (FcγRIIIa). For example, co-administration ofa therapeutic antibody and a compound that blocks an inhibitory receptoror stimulates an activating receptor on NK cells allows a lower dose ofthe therapeutic antibody to be used. Such antibodies can be used at a20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or lower dose than therecommended dose in the absence of the compound.

In addition, the invention provides a method for determining atherapeutically-effective, reduced dose of a therapeutic antibody, e.g.,an antibody bound by CD16, the method comprising i) co-incubating afirst concentration of the therapeutic antibody with target cells and NKcells, and in the absence of a compound that blocks an inhibitoryreceptor or stimulates an activating receptor on NK cells; ii)co-incubating a second, lower concentration of the therapeutic antibodywith target cells, with NK cells, and in the presence of a compound thatblocks an inhibitory receptor or stimulates an activating receptor on NKcells; iii) determining if the depletion of target cells observed instep ii) is as great as the depletion observed in step i). If it isobserved that step ii) is as efficacious as step i), then the relativeconcentrations of the compound and the therapeutic antibody can bevaried, and depletion observed, in order to identify differentconditions that would be suitable for use in a given patient, e.g.,maximizing target cell depletion, lowered dose of therapeutic antibody,or lowered dose of the compound, depending on the particular needs ofthe patient.

In a particular aspect, the present invention provides a method oftreatment of a disease in a human subject in need thereof, comprising:a) administering to said subject a compound that blocks an inhibitoryreceptor or stimulates an activating receptor of an NK cell; and, b)administering to said subject a therapeutic antibody that can be boundby CD16.

In one embodiment, the therapeutic antibody and compound areadministered into the subject simultaneously. In another embodiment, thecompound is administered to the subject within one week, within 4 days,within 3 days or on the same day (e.g., within about 24 hours) of theadministration of the therapeutic antibody. In another embodiment, thedisease is a cancer, infectious or immune disease.

In one embodiment, the method further comprising an additional step inwhich the activity or number of NK cells in the subject is assessedprior or subsequent to the administration of the compound. In anotherembodiment, the additional step involves i) obtaining NK cells from thesubject prior to the administration; ii) incubating the NK cells in thepresence of one or more target cells that are recognized by thetherapeutic antibody, in the presence or absence of the compound; andiii) assessing the effect of the compound on the ability of the NK cellsto deplete the target cells; wherein a detection that the compoundenhances the ability of the NK cells to deplete the target cellsindicates that the compound is suitable for use in the method, and thatthe method is suitable for use with the subject.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutic antibody, e.g., that can be boundby CD16, a compound that blocks an inhibitory receptor or stimulates anactivating receptor of NK cells, and a pharmaceutically acceptablecarrier. In another aspect, the present invention provides a kitcomprising a therapeutic antibody, e.g., that can be bound by CD16, andone or more compounds that block an inhibitory receptor or stimulate anactivating receptor of NK cells.

For any of the above-mentioned methods, compositions, or kits, in oneembodiment the therapeutic antibody has a human IgG1 or an IgG3 Feportion. In another embodiment, the compound is an antibody or afragment thereof. In another embodiment, the therapeutic antibody is amonoclonal antibody or fragment thereof. In another embodiment, thetherapeutic antibody is not conjugated with a radioactive or toxicmoiety. In another embodiment, the compound inhibits an inhibitoryreceptor of an NK cell. In another embodiment, the compound stimulatesan activating receptor of an NK cell. In another embodiment, thecompound is a human, humanized or chimeric antibody, or a fragmentthereof. In one embodiment, the therapeutic antibodies or compounds canbe antibody fragments or derivatives such as, inter alia, a Fabfragment, a Fab′2 fragment, a CDR and a ScFv.

In one embodiment, the therapeutic antibody is a human, humanized orchimeric antibody or a fragment thereof. In another embodiment, thetherapeutic antibody is rituximab or Campath. In another embodiment, theantibody is rituximab, and said antibody is administered at a dosage ofless than 375 mg/m² per week. In another embodiment, the antibody isCampath, and the antibody is administered at a dosage of less than 90 mgper week.

In one embodiment, the compound binds at least one of NKG2, KIR2DL orKIR3DL human receptors, and inhibits the related NKG2, KIR2DL- orKIR3DL-mediated inhibition of NK cell cytotoxicity. In anotherembodiment, the compound blocks an inhibitory receptor of an NK cellselected from the group consisting of KIR2DL1, KIR2DL2/3, KIR2DL4,KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2CNKG2E and LILRB5. In another embodiment, the compound binds a commondeterminant of KIR2DL human receptors and inhibits KIR₂DL-mediatedinhibition of NK cell cytotoxicity. In another embodiment, the compoundbinds a common determinant of KIR2DL1, KIR2DL2, and KIR2DL3 humanreceptors and inhibits KIR2DL1-, KIR2DL2-, and KIR2DL3-mediatedinhibition of NK cell cytotoxicity. In another embodiment, the compoundinhibits the binding of a HLA-C allele molecule having a Lys residue atposition 80 to a human KIR2DL1 receptor, and the binding of a HLA-Callele molecule having an Asn residue at position 80 to human KIR2DL2and KIR2DL3 receptors. In another embodiment, the compound binds to thesame epitope as monoclonal antibody DF200 produced by hybridoma DF200.In another embodiment, the compound competes with monoclonal antibodyDF200 produced by hybridoma DF200 for binding to a KIR receptor at thesurface of a human NK cell. In another embodiment, the compound ismonoclonal antibody DF200 produced by hybridoma DF200 or a fragmentthereof.

In one embodiment, the compound binds to a receptor selected from thegroup consisting of NKp30, NKp44, NKp46, and NKG2D. In anotherembodiment, the compound is derived from or competes with a monoclonalantibody selected from the group consisting of AZ20, A76, Z25, Z231, andBAB281.

In another aspect, the present invention provides a method of selectinga compound for administration in conjunction with a therapeuticantibody, said method comprising: i) providing a test compound thatinhibits an inhibitory receptor or stimulates an activating receptor ofNK cells; ii) incubating the therapeutic antibody with target cellsspecifically recognized by the therapeutic antibody in the presence ofNK cells and in the presence or absence of the test compound; and iii)assessing the effect of the compound on the ability of the NK cells todeplete the target cells; wherein a detection that the compound enhancesthe ability of the NK cells to deplete the target cells indicates thatthe compound is suitable for use in the method.

In one embodiment, the compound enhances the ability of the therapeuticantibody to destroy the target cells by 50%, 60%, 70%, 80%, 90%, 100%,200%, 300%, 400%, 500%, or more. In another embodiment, the compound isselected from the group consisting of an antibody, an antibody fragment,a monoclonal antibody, a fragment of a monoclonal antibody, a humanizedantibody, a chimeric antibody, and a human antibody. In anotherembodiment, the target cells are cancer cells, virally infected cells,or cells underlying an autoimmune disorder. In another embodiment, thetherapeutic antibody is rituximab or CAMPATH.

In another aspect, the present invention provides a method of increasingthe efficiency of a treatment involving the administration of atherapeutic antibody that can be bound by CD16 in a subject, said methodcomprising administering to said subject prior to, simultaneously with,or after the administration of said therapeutic antibody, atherapeutically-effective amount of a compound that blocks an inhibitoryreceptor or stimulates an activating receptor of an NK cell. In oneembodiment, the compound increases the efficiency of the treatment byenhancing ADCC in said subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D: Monoclonal antibody DF200 binds a common determinant ofvarious human KIR2DL receptors. One of the monoclonal antibodies, theDF200 mAb, was found to react with various members of the KIR family,including: KIR2DL1 and KIR2DL2/3. Regarding the staining of NK cellswith DF200 mAb, both KIR2DL1+ and KIR2DL2/3+ cells were stainedbrightly. FIG. 1A: Clone CP11 KIR2DL1+DF200; FIG. 1B: Clone CP11KIR2DL1+ANTIKIR2DL1; FIG. 1C: Clone CP502 KIR2DL3+DF200; FIG. 1D: CloneCP502 KIRDL3+ANTIKIR2DL2/3.

FIG. 2: Reconstitution of lysis with anti-KIR2DL mAb (monoclonalantibody) on C1R Cw4 target at effector/target ratio of 4/1. Monoclonalantibody DF200 inhibits KIR2DL-mediated inhibition of KIR2DL1 positiveNK cell cytotoxicity (reconstitute lysis) on Cw4 positive target cells.

FIG. 3: Enhancement of ADCC mediated by Rituxan of a KIR2DL1 positive NKclone on a Cw4 positive EBV cell line by blocking KIR/HLA interaction.NK clone cytolysis bearing KIR2DL1 is tested against a Cw4 positive EBVtransformed (CD20 positive) target cell line at various effector/targetratio (from 1 to 4) in the presence of 5 μg/ml anti CD20 antibody(Rutixan) and 10 μg/ml EB6 antibody (anti KIR2DL1); Rituxan alone; EB6alone; or without any antibody. ADCC is greatly enhanced in the presenceof anti KIR2DL1 antibody (EB6).

FIG. 4: Enhancement of ADCC mediated by Campath of a KIR2DL1 positive NKclone on a Cw4 positive EBV cell line by blocking KIR/HLA interaction.NK clone cytolysis bearing KIR2DL1 is tested against a Cw4 positive EBVtransformed (CD20 positive) target cell line in the presence of Campathand 100 μg/ml EB6 antibody (anti KIR2DL1); Campath alone; EB6 alone; orwithout any antibody. ADCC is greatly enhanced in the presence of theanti KIR2DL1 antibody (EB6).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for increasing the efficiency oftherapeutic antibodies. The invention more specifically discloses thatthe use of a compound, preferably an antibody or a fragment thereof,that potentiates NK cells, preferably by blocking an inhibitory receptoror activating an activating receptor of an NK cell, can significantlyincrease the efficiency of therapeutic antibodies. Indeed, the inventorsdemonstrate that the efficiency of multiple therapeutic antibodies canbe greatly enhanced by the co-administration of an antibody directedagainst an NK cell receptor; e.g., an inhibitory receptor.

Therefore, the invention concerns a method of treatment of a disease ina subject in need thereof comprising:

a) administering to said subject a compound, preferably an antibody or afragment thereof, that blocks an inhibitory receptor or stimulates anactivating receptor of an NK cell; and,

b) administering to said subject a therapeutic antibody. Saidtherapeutic antibody can be bound by CD16 of NK cells, preferablythrough its Fc region.

Preferably, said therapeutic antibody has a human IgG1 or an IgG3 Fcportion, particularly a monoclonal antibody or a fragment thereof,further preferably a humanized, human or chimeric antibody or a fragmentthereof, for instance rituximab.

It is intended that compounds, preferably antibodies or a fragmentthereof, that block the inhibitory receptor of an NK cell can beadministered to the subject before, simultaneously with or, after theadministration of the therapeutic antibody. The way of administration ofthe different antibodies depends on their bioavailability andphamacokinetics. Preferably, the therapeutic antibody is administratedwithin a week to the administration of the compounds, preferablyantibodies or a fragment thereof, that block the inhibitory receptor ofan NK cell, more preferably within the 5 or 2 days period. Preferably,the therapeutic antibody is administrated before or simultaneously withthe compounds, preferably antibodies or a fragment thereof, that blockthe inhibitory receptor of an NK cell.

In a further aspect, the invention concerns a method of increasing ADCCin a subject receiving a therapeutic antibody treatment, said methodcomprising administering to said subject prior to, simultaneously orafter the administration of said therapeutic antibody an amountsufficient to increase ADCC of a compound, preferably an antibody or afragment thereof, that blocks the inhibitory receptor of an NK cell.Said therapeutic antibody can be bound by CD16 on NK cells, preferablythrough its Fc region. Preferably, said therapeutic antibody has a humanIgG1 or an IgG3 Fc portion, particularly a monoclonal antibody or afragment thereof, further preferably a human, humanized or chimericantibody or a fragment thereof, for instance rituximab.

In an additional aspect, the invention concerns a method of increasingthe efficiency of a therapeutic antibody treatment in a subject, saidmethod comprising administering to said subject prior to, simultaneouslyor after the administration of said therapeutic antibody an amount of acompound, preferably an antibody or a fragment thereof, that blocks theinhibitory receptor of an NK cell, sufficient to increase the efficiencyof said therapeutic antibody. Said therapeutic antibody can be bound byCD16, preferably through its Fc region. Preferably, said therapeuticantibody has a human IgG1 or IgG3 Fc portion, particularly a monoclonalantibody or a fragment thereof, further preferably a human, humanized orchimeric antibody or a fragment thereof, for instance rituximab.

DEFINITIONS

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

As used herein, “NK” cells refers to a sub-population of lymphocytesthat is involved in non-conventional immunity. NK cells can beidentified by virtue of certain characteristics and biologicalproperties, such as the expression of specific surface antigensincluding CD16, CD56 and/or CD57, the absence of the alpha/beta orgamma/delta TCR complex on the cell surface, the ability to bind to andkill cells that fail to express “self” MHC/HLA antigens by theactivation of specific cytolytic enzymes, the ability to kill tumorcells or other diseased cells that express a ligand for NK activatingreceptors, and the ability to release protein molecules called cytokinesthat stimulate or inhibit the immune response. Any of thesecharacteristics and activities can be used to identify NK cells, usingmethods well known in the art.

The term “antibody,” as used herein, refers to polyclonal and monoclonalantibodies. Depending on the type of constant domain in the heavychains, antibodies are assigned to one of five major classes: IgA, IgD,IgE, IgG, and IgM. Several of these are further divided into subclassesor isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplaryimmunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids that is primarily responsible forantigen recognition. The terms variable light chain (V_(L)) and variableheavy chain (V_(H)) refer to these light and heavy chains respectively.The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are termed “alpha,” “delta,” “epsilon,”“gamma” and “mu,” respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG and/or IgM are the preferred classes of antibodiesemployed in this invention, with IgG being particularly preferred,because they are the most common antibodies in the physiologicalsituation, because they are most easily made in a laboratory setting,and because IgGs are specifically recognized by Fc gamma receptors.Preferably the antibody of this invention is a monoclonal antibody.Particularly preferred are humanized, chimeric, human, orotherwise-human-suitable antibodies.

Within the context of this invention, the term “therapeutic antibody orantibodies” designates more specifically any antibody that functions todeplete target cells in a patient. In particular, therapeutic antibodiesspecifically bind to antigens present on the surface of the targetcells, e.g., tumor specific antigens present predominantly orexclusively on tumor cells. Preferably, therapeutic antibodies includehuman Fc portions, or are capable of interacting with human Fcreceptors. Therapeutic antibodies can target cells by any means, e.g.,ADCC or otherwise, and can be “naked,” i.e., with no conjugatedmoieties, or they can be conjugated with compounds such as radioactivelabels or toxins.

The term “specifically binds to” means that an antibody can bindpreferably in a competitive binding assay to the binding partner, e.g.,an activating NK receptor such as NKp30, NKp44, or NKp46, or a human Fcgamma receptor, as assessed using either recombinant forms of theproteins, epitopes therein, or native proteins present on the surface ofisolated NK or relevant target cells. Competitive binding assays andother methods for determining specific binding are further describedbelow and are well known in the art.

A “human-suitable” antibody refers to any antibody, derivatizedantibody, or antibody fragment that can be safely used in humans for,e.g., the therapeutic methods described herein. Human-suitableantibodies include all types of humanized, chimeric, or fully humanantibodies, or any antibodies in which at least a portion of theantibodies is derived from humans or otherwise modified so as to avoidthe immune response that is provoked when native non-human antibodiesare used.

By “immunogenic fragment”, it is herein meant any polypeptidic orpeptidic fragment which is capable of eliciting an immune response suchas: (i) the generation of antibodies binding said fragment and/orbinding any form of the molecule comprising said fragment, including themembrane-bound receptor and mutants derived therefrom, (ii) thestimulation of a T-cell response involving T-cells reacting to thebi-molecular complex comprising any MHC molecule and a peptide derivedfrom said fragment, (iii) the binding of transfected vehicles such asbacteriophages or bacteria expressing genes encoding mammalianimmunoglobulins. Alternatively, an immunogenic fragment also refers toany construction capable to elicit an immune response as defined above,such as a peptidic fragment conjugated to a carrier protein by covalentcoupling, a chimeric recombinant polypeptide construct comprising saidpeptidic fragment in its amino acid sequence, and specifically includescells transfected with a cDNA of which sequence comprises a portionencoding said fragment.

For the purposes of the present invention, a “humanized” antibody refersto an antibody in which the constant and variable framework region ofone or more human immunoglobulins is fused with the binding region,e.g., the CDR, of an animal immunoglobulin. Such humanized antibodiesare designed to maintain the binding specificity of the non-humanantibody from which the binding regions are derived, but to avoid animmune reaction against the non-human antibody.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity. In preferred embodiments of the presentinvention, the chimeric antibody nevertheless maintains the Fc region ofthe immunoglobulin, preferably a human Fc region, thereby allowinginteractions with human Fc receptors on the surface of target cells.

Within the context of this invention, “potentiated,” “active,” or“activated” NK cells designate biologically active NK cells, moreparticularly NK cells having the capacity of lysing target cells. Forinstance, an “active” NK cell is able to kill cells that express an NKactivating receptor-ligand and fails to express “self” MHC/HLA antigens(KIR-incompatible cells). Examples of suitable target cells for use inredirected killing assays are P815 and K562 cells, but any of a numberof cell types can be used and are well known in the art (see, e.g.,Sivori et al. (1997) J. Exp. Med. 186: 1129-1136; Vitale et al. (1998)J. Exp. Med. 187: 2065-2072; Pessino et al. (1998) J. Exp. Med. 188:953-960; Neri et al. (2001) Clin. Diag. Lab. Immun. 8:1131-1135).“Potentiated,” “active,” or “activated” cells can also be identified byany other property or activity known in the art as associated with NKactivity, such as cytokine (e.g., IFN-γ and TNF-α) production ofincreases in free intracellular calcium levels. For the purposes of thepresent invention, “potentiated,” “active,” or “activated” NK cellsrefer particularly to NK cells in vivo that are not inhibited viastimulation of an inhibitory receptor, or in which such inhibition hasbeen overcome, e.g., via stimulation of an activating receptor.

As used herein, the term “activating NK receptor” refers to any moleculeon the surface of NK cells that, when stimulated, causes a measurableincrease in any property or activity known in the art as associated withNK activity, such as cytokine (for example IFN-γ and TNF-α) production,increases in intracellular free calcium levels, the ability to targetcells in a redirected killing assay as described, e.g., elsewhere in thepresent specification, or the ability to stimulate NK cellproliferation. The term “activating KIR receptor” includes but is notlimited to NKp30, NKp44, NKp46, NKG2D, IL-12R, IL-15R, IL-18R andIL-21R. The term “activating NK receptor” as used herein excludes theIL-2 receptor (IL-2R). Methods of determining whether an NK cell isactive or proliferating or not are described in more detail below andare well known to those of skill in the art.

As used herein, the term “inhibiting” or “inhibitory” NK receptor”refers to any molecule on the surface of NK cells that, when stimulated,causes a measurable decrease in any property or activity known in theart as associated with NK activity, such as cytokine (e.g., IFN-γ andTNF-α) production, increases in intracellular free calcium levels, orthe ability to lyse target cells in a redirected killing assay asdescribed, e.g., elsewhere in the present specification. Examples ofsuch receptors include KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B,KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C NKG2E and LILRB5.Methods of determining whether an NK cell is active or not are describedin more detail below and are well known to those of skill in the art.

In the present invention, the term “block an inhibitory receptor orstimulates an activating receptor of an NK cell” refers to the abilityof certain compounds, preferably antibodies, fragments or derivativesthereof; to preferably directly interact with at least one inhibitory oractivating NK cell receptor, e.g., KIR, NKG2A/C, NKp30, NKp44, NKp46 andothers listed herein, and either neutralizing inhibitory signals of thereceptor (in the case of inhibitory receptors) or stimulate signallingfrom the receptor (in the case of activating receptors). With inhibitoryreceptors, preferably the compound, preferably an antibody or a fragmentthereof, is able to block the interaction between HLA and the receptor.When the compound is an antibody, the antibodies may by polyclonal or,preferably, monoclonal. They may be produced by hybridomas or byrecombinant cells engineered to express the desired variable andconstant domains. The antibodies may be single chain antibodies or otherantibody derivatives retaining the antigen specificity and the lowerhinge region or a variant thereof such as a Fab fragment, a Fab′2fragment, a CDR and a ScFv. These may be polyfunctional antibodies,recombinant antibodies, humanized antibodies, or variants thereof.

Within the context of this invention a “common determinant” designates adeterminant or epitope that is shared by several members of a group ofrelated receptors, e.g., the human KIR2DL receptor group. Thedeterminant or epitope may represent a peptide fragment or aconformational epitope shared by said members. In a specific embodiment,the common determinant comprises an epitope recognized by monoclonalantibody DF200, NKVSF1 or EB6.

Within the context of this invention, the term antibody that “binds” acommon determinant designates an antibody that binds said determinantwith specificity and/or affinity, e.g., that essentially does not bindwith high affinity or with specificity other unrelated motifs ordeterminant or structures at the surface of human NK cells. Moreparticularly, the binding of a monoclonal antibody according to thisinvention to said determinant can be discriminated from the binding ofsaid antibody to another epitope or determinant.

Compounds, preferably antibodies, capable of binding to NK cellinhibitory receptors and prevent their stimulation are thus“neutralizing” or “inhibitory” compounds, preferably antibodies, in thesense that they block, at least partially, the inhibitory signallingpathway mediated by an NK cells inhibitory receptor, i.e., KIR orNKG2A/C receptors. More importantly, this inhibitory activity can bedisplayed with respect to several types of KIR or NKG2A/C receptors, sothat these compounds, preferably antibodies, may be used in varioussubjects with high efficacy.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all.

Within the context of the present invention, a subject or patientincludes any mammalian subject or patient, more preferably a humansubject or patient.

Therapeutic Antibodies

The present invention deals with the use of NK cell potentiatingcompounds in conjunction with therapeutic antibodies. Any of a largevariety of therapeutic antibodies can be used in the present invention.Essentially, any therapeutic antibody, whether “naked” or conjugatedwith a radiolabel, toxin, or other moiety, or whether full length or afragment; or whether a true antibody or a modified derivative of anantibody, can be used. Preferably, the methods are used to enhance theefficacy of therapies in which NK cell activity plays a role—notnecessarily exclusive—in the efficacy of administered therapeuticantibodies, and also preferably the antibodies or fragments willnaturally include, or will be modified to include, a human Fc region orother domain that allows specific recognition of the antibody by humanFc receptors, e.g., Fc gamma receptors.

The present compounds can be used to enhance the ability of therapeuticantibodies to deplete target cells that express an antigen that isspecifically recognized by the therapeutic antibodies. Accordingly, anydisease or condition that is caused or exacerbated at least in part bycells that can be targeted by a therapeutic antibody can be treatedusing the herein-described methods. Specific examples of target cellsinclude tumor cells, virus-infected cells, allogenic cells, pathologicalimmunocompetent cells (e.g., B lymphocytes, T lymphocytes,antigen-presenting cells, etc.) involved in allergies, autoimmunediseases, allogenic reactions, etc., or even healthy cells (e.g.,endothelial cells in an anti-angiogenic therapeutic strategy). Mostpreferred target cells within the context of this invention are tumorcells and virus-infected cells. The therapeutic antibodies may, forinstance, mediate a cytotoxic effect or cell lysis, particularly byantibody-dependent cell-mediated cytotoxicity (ADCC).

ADCC requires leukocyte receptors for the Fc portion of IgG (FcγR),whose function is to link the IgG-sensitized antigens to FcγR-bearingcytotoxic cells and to trigger the cell activation machinery. Therefore,the therapeutic antibody is capable of forming an immune complex. Forexample, an immune complex can be a tumor target covered by therapeuticantibodies. More particularly, the antibody can be bound by CD16,preferably through its Fc region. Determining whether a therapeuticantibody binds an Fcγ receptor such as CD16 can be assessed by anysuitable manner, for example by determining binding to a recombinantproduced CD16 polypeptide or fragment thereof, optionally immobilized ona support, or for example by determining binding of the therapeuticantibody to a cell which known or suspected to express CD16.

The therapeutic antibodies may by polyclonal or, preferably, monoclonal.They may be produced by hybridomas or by recombinant cells engineered toexpress the desired variable and constant domains. The antibodies may besingle chain antibodies or other antibody derivatives retaining theantigen specificity and the lower hinge region or a variant thereof.These may be polyfunctional antibodies, recombinant antibodies,humanized antibodies, fragments or variants thereof. Said fragment or aderivative thereof is preferably selected from a Fab fragment, a Fab′2fragment, a CDR and a ScFv. Preferably a fragment is an antigen-bindingfragment. Therapeutic antibodies which comprise an antibody fragment mayalso include but are not limited to bispecific antibodies; one example asuitable bispecific antibody comprises an antigen binding regionspecific for CD16 and an antigen binding region specific for a tumorantigen. Other antibody formats comprising fragments include recombinantbispecific antibody derivatives combining the binding regions of twodifferent antibodies on a single polypeptide chain, also referred to asBiTE™ (Kufer P, et al TRENDS in Biotechnology 2004; 22 (5): 238-244; andBaeuerle et al, Current Opinion in Molecular Therapeutics 2003; 5(4):413-419, the disclosures of which are incorporated herein by reference.

Therapeutic antibodies are generally specific for surface antigens,e.g., membrane antigens. Most preferred therapeutic antibodies arespecific for tumor antigens (e.g., molecules specifically expressed bytumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25,MUC-1, CEA, KDR, αVβ3, etc., particularly lymphoma antigens (e.g.,CD20). The therapeutic antibodies have preferably human or non humanprimate IgG1 or IgG3 Fc portion, more preferably human IgG1.

In one embodiment, the antibodies will include modifications in their Fcportion that enhances the interaction of the antibody with NK cellsduring ADCC. Such modified therapeutic antibodies (“altered antibodies”)generally comprise modifications preferably, in the Fc region thatmodify the binding affinity of the antibody to one or more FcγR. Methodsfor modifying antibodies with modified binding to one or more FcγR areknown in the art, see, e.g., PCT Publication Nos. WO 2004/016750(International Application PCT/US2003/025399), WO 99/158572, WO99/151642, WO 98/123289, WO 89/107142, WO 88/107089, and U.S. Pat. Nos.5,843,597 and 5,642,821, each of which is incorporated herein byreference in their entirety.

Therapeutic antibodies identified herein, such as D2E7 (CambridgeAntibody Technology Group, plc (Cambridge, UK)/BASF (Ludwigshafen,Germany)) used to treat rheumatoid arthritis, or Infliximab (Centocor,Inc., Malvern, Pa.; used to treat Crohn's disease and rheumatoidarthritis), or the antibodies disclosed in International PatentApplication PCT/US2003/025399 (which is hereby incorporated by referencein its entirety) can be modified as taught in the above and belowidentified applications and used for the treatment of diseases for whichsuch antibodies are typically used. In some embodiments, the inventionprovides altered antibodies that have altered affinity, either higher orlower affinity, for an activating FcγR, e.g., FcγRIII. In certainpreferred embodiments, altered antibodies having higher affinity forFcγR are provided. Preferably such modifications also have an alteredFe-mediated effector function.

Modifications that affect Fc-mediated effector function are well knownin the art (See, e.g., U.S. Pat. No. 6,194,351, which is incorporatedherein by reference in its entirety). The amino acids that can bemodified include but are not limited to proline 329, proline 331, andlysine 322. Proline 329 and/or 331 and lysine 322 can, preferably bereplaced with alanine, however, substitution with any other amino acidis also contemplated. See International Publication No.: WO 00/142072and U.S. Pat. No. 6,194,551 which are incorporated herein by referencein their entirety. Thus, modification of the Fc region can comprise oneor more alterations to the amino acids found in the antibody Fc region.Such alterations can result in an antibody with an alteredantibody-mediated effector function, an altered binding to other Fcreceptors (e.g., Fc activation receptors), an altered ADCC activity, analtered Clq binding activity, an altered complement dependentcytotoxicity activity, or any combination thereof.

In one embodiment, the antibody is specifically recognized by an Fcgamma receptor such as FCGR3A (also called CD16, FCGR3, Immunoglobulin GFc Receptor III; IGFR₃, Receptor for Fc Fragment of IgG, Low AffinityIxia; see, e.g., OMIM 146740), FCGR2A (also called CD32, CDw32, Receptorfor Fe Fragment of IgG, Low Affinity IIa, FCG2, Immunoglobulin G FeReceptor II; see, e.g., OMIM 146790); FCGR2B (also called CD32, Receptorfor Fc Fragment of IgG, Low Affinity IIb; FCGR2B, FC-Gamma-RIIB; see,e.g., OMIM 604590), FCG1RA (also called CD64; Receptor for Fc Fragmentof IgG, High affinity Ia; IGFR1; see, e.g., OMIM 146760); FCGR1 fragmentof IgG, High affinity Ic, Immunoglobulin G Fc receptor IC, IGFRC; see,e.g., OMIM 601503); or FCGR1B (also called CD64, Receptor for FcFragment of IgG, High affinity Ib; Immunoglobulin G Fc Receptor IB;IGFRB; see, e.g., OMIM 601502).

Typical examples of therapeutic antibodies of this invention arerituximab, alemtuzumab and trastuzumab. Such antibodies may be usedaccording to clinical protocols that have been authorized for use inhuman subjects. Additional specific examples of therapeutic antibodiesinclude, for instance, epratuzumab, basiliximab, daclizumab, cetuximab,labetuzumab, sevirumab, tuvurimab, palivizumab, infliximab, omalizumab,efalizumab, natalizumab, clenoliximab, etc. Optionally, when a compoundthat stimulates an activating receptor of an NK cell is a cytokine, thetherapeutic antibody is an antibody other than rituximab or herceptin,or optionally other than an anti-CD20 or anti-HER2/neu antibody. Otherexamples of preferred therapeutic antibodies for use in accordance withthe invention include anti-ferritin antibodies (US Patent Publicationno. 2002/0106324), anti-p140 and anti-sc5 antibodies (WO 02/50122), andanti-KIR (killer inhibitory receptor) antibodies (The KIR receptors aredescribed in Carrington and Norman, The KIR Gene Cluster, May 3, 2003,available at: worldwide web site ncbi.nlm.nih.gov/books), thedisclosures of each of the above reference being incorporated herein byreference. Other examples of therapeutic antibodies are listed in thefollowing table, any of which (and others) can be used in the presentmethods. It will be appreciated that, regardless of whether or not theyare listed in the following table or described elsewhere in the presentspecification, any antibody that can deplete target cells, preferably byADCC, can benefit from the present methods, and that the following Table1 is non exhaustive, neither with respect to the antibodies listedtherein, nor with respect to the targets or indications of theantibodies that are listed.

TABLE 1 Therapeutic antibodies Ab specificity DCI Commercial nameTypical Indications Anti-CD20 rituximab MabThera ®, NHL B Rituxan ®Anti-CD20 Zevalin NHL Anti-CD20 Bexocar NHL Anti-CD52 alemtuzumabCAMPATH-1H ® CLL, allograft Anti-CD33 SMART-M195 AML Anti-CD33 Zamyl ™Acute myeloid Leukemia Anti-HLA-DR SMART-ID10 NHL antigen Anti-HLA-DRRemitogen ™ NHL B Anti-CD22 epratuzumab LymphoCide ™ NHL B Anti-HER2MDX-210 Prostate and other cancers Anti-erbB2 trastuzumab Herceptin ®,Metastatic breast cancer (HER-2/neu) Anti-CA125 OvaRex Ovarian cancerAnti-MUC1 TriAb Metastatic breast cancer Anti-MUC1 BravaRex Metastaticcancers Anti-PEM antigen Theragyn, Therex Ovarian cancer, breast cancerAnti-CD44 bivatuzumab Head and neck cancer Anti-gp72 MAb, colorectalcancer idiotypic 105AD7 Anti-EpCAM Anti-EpCAM; IS-IL2 cancer MT201Anti-VEGF MAb-VEGF metastatic NSCLC, colorectal cancer Anti-CD18 AMD Fabage-related macular degeneration Anti-CD18 Anti-CD18 Myocardialinfarction Anti-VEGF IMC-1cl I colorectal cancer receptor anti-nuC242nuC242-DMI Colorectal, gastric, and pancreatic cancer Anti-EGFR MAb425cancer Anti-EGFR ABX-EGF Cancer Anti-EGFR cetuximab ENT and colorectalCancers (HER-1, erbB1) Anti-MUC-1 Therex ® Breast and epithelial cancersAnti-CEA CEAVac Colorectal cancer Anti-CEA labetuzumab CEA-Cide ™ Solidtumors Anti-αVβ3 Vitaxin Leiomyosarcoma, colorectal and other cancers(anti-angiogenic) Anti-KDR Cancers (anti-angiogenic) (VEGFR2) anti-VRSfusion palivizumab Synagis ® Viral diseases protein Idem Numax ™ IdemCMV sevirumab Protovir CMV Infection HBs tuvirumab Ostavir ™ Hepatitis BAnti-CD25 basiliximab Simulect ® Prevention/treatment allograftrejection Anti-CD25 daclizumab Zenapax ® Prevention/treatment allograftrejection anti-TNF-α infliximab Remicade ™ Crohn's disease, rheumatoidarthritis anti-CD80 IDEC-114 psoriasis anti-IgE E-26 Allergic asthma andrhinitis anti-IgE omalizumab Xolair ™ Asthma anti-IgE Rhu-mAbAllergy/asthma E25 anti-integrin αL efalizumab Xanelim ™ psoriasis(CD11a, LFA-1) Anti-beta 2 LDP-01 Stroke, allograft rejection integrinanti-integrin αL anti-CD11a psoriasis (CD11a, LFA-1) anti-CD4 keliximabGVHD, psoriasis siplizumab MEDI-507 Anti-CD4 OKT4A Allograft rejectionAnti-CD3 OKT3 Allograft rejection Anti-CD3 SMART- Autoimmune disease,allograft aCD3 rejection, psoriasis Anti-CD64 anemia anti-CD147 GvHDanti-integrin α4 natalizumab Antegren ® Multiple Sclerosis, Crohn's(α4β1-α4β7) Disease Anti-integrin β7 Crohn's Disease, ulcerative colitisAlpha 4 beta 7 LDP-02 Ulcerative colitis Anti-HLA-DR10 Oncolym NHL betaAnti-CD3 Nuvion T cell malignancies Anti-GD2 Trigem Metastatic melanomaand small ganglioside cell lung cancer Anti-SK-1 antigen Colorectal andpancreatic carcinoma anti-CD4* clenoliximab anti-IL-8 ABX-IL8 psoriasisAnti-VLA-4 Antegren MS Anti-CD40L Antova SLE, allograft rejectionAnti-CD40L IDEC-131 MS, SLE Anti-E-selectin CDP850 psoriasisAnti-CD11/CD18 Hu23F2G MS, stroke Anti-ICAM-3 ICM3 psoriasis Anti-CBLABX-CBL GVHD Anti-CD147 Anti-CD23 IDEC-152 Asthma, allergies Anti-CD25Simulect Allograft rejection Anti-T1-ACY ACY-110 Breast cancer Anti-TTSTTS-CD2 Pancreatic, renal cancer Anti-TAG72 AR54 Breast, ovarian, lungcancer Anti-CA19.9 GivaRex Colorectal, pancreatic, gastric Anti-PSAProstaRex Prostate cancer Anti-HMFG1 R1550 Breast, gastric cancerpemtumomab Theragyn Gastric, ovarian cancer Anti-hCG CTP-16, Multiplecancers CTP-21 Anti collagen HU177; Multiple cancers Types 1-V HUIV26;XL313 Anti-CD46 Crucell/J&J Multiple cancers Anti-17A-1 EdrecolomabPanorex Colorectal cancer Anti-HM1.24 AHM Multiple myeloma Anti-CD38Anti-CD38 Multiple myeloma Anti-IL15 HuMax Lymphoma Receptor lymphomaAnti-IL6 B-E8 Lymphoma Anti-TRAIL-R1 TRM-1 Multiple cancers Anti-VEGF2Multiple cancers Anti-BlyS Lymphostat Multiple cancers Anti-SCLC, CEAPentacea Lung cancer and DTPA Anti-CD52 CAMPATH Leukemia, LymphomaAnti-Lewis Y IGN311 Epithelial cancers antigen Anti-VE cadherin E4G10Multiple cancers Anti-CD56 BB10901, Colorectal, lung cancer huN901DC1Anti- Cantuzumab Colorectal, lung, pancreatic mertansine/mucine cancerAnti-AFP AFP-cide Liver cancer Anti-CSAp Mu-9 Colorectal cancerAnti-CD30 MDX-060 Melanoma, Hodgkins Disease Anti-PSMA MDX-070 Prostatecancer Anti-CD15 MDX-11 Leukemia Anti-TAG72 MDX-020 Colorectal cancerAnti-CD19, CD3 MT103 Lymphoma bispecific Anti-mesothelin SS1-PE38 Brainand ovarian cancer, antigen mesothelioma Anti-DNA and Cotara Colorectal,pancreatic, sarcoma, histones brain and other cancers Anti-a5B1 integrinAnti-a5 B1 Multiple cancers Anti-p97 SGN17/19 Melanoma Anti-CD5 GenimuneLeukemia, lymphoma

Compounds Regulating NK Cell Activity

NK cell activity is regulated by a complex mechanism that involves bothstimulating and inhibitory signals. Accordingly, effective NKcell-mediated therapy can be achieved both by a stimulation of thesecells or a neutralization of inhibitory signals. It will be appreciatedthat any compound that has the effect of blocking, inhibiting, orotherwise downregulating an inhibitory receptor of an NK cell, or ofactivating, stimulating, or otherwise promoting the activity orexpression of an activating receptor of an NK cell, can be used. Thisincludes compounds such as cytokines, as well as small molecules,polypeptides, and antibodies that can bind to NK cell receptors anddirectly inhibit or stimulate them. It will also be appreciated that themechanism by which the receptors are blocked or stimulated is notcritical to the advantages provided by the invention. For example, thecompounds can increase the expression of an activating receptor, orinhibit the expression of an inhibitory receptor, the compounds canprevent the interaction between a ligand and an inhibitory receptor orenhance the interaction between a ligand and an activating receptor, orthe compounds can bind directly to the receptors and inhibit them (inthe case of inhibitory receptors) or activate them (in the case ofactivating receptors). The critical parameter is the effect that thecompounds have on the ability of therapeutic antibodies to deplete theirtarget cells in vivo.

Any inhibitory receptor on the surface of an NK cell can be targeted bythe present compounds. NK cells are negatively regulated by majorhistocompatibility complex (MHC) class I-specific inhibitory receptors(Kärre et al., 1986; Öhlén et al, 1989; the disclosures of which areincorporated herein by reference). These specific receptors bind topolymorphic determinants of major histocompatibility complex (MHC) classI molecules or HLA and inhibit natural killer (NK) cell lysis. Inhumans, a family of receptors termed killer Ig-like receptors (KIRs)recognize groups of HLA class I alleles.

There are several groups of KIR receptors, including KIR2DL, KIR2DS,KIR3DL and KIR3DS. KIR receptors having two Ig domains (KIR2D) identifyHLA-C allotypes: KIR2DL2 (formerly designated p58.1) or the closelyrelated gene product KIR2DL3 recognizes an epitope shared by group 2HLA-C allotypes (Cw1, 3, 7, and 8), whereas KIR2DL1 (p58.2) recognizesan epitope shared by the reciprocal group 1 HLA-C allotypes (Cw2, 4, 5,and 6). The recognition by KIR2DL1 is dictated by the presence of a Lysresidue at position 80 of HLA-C alleles. KIR2DL2 and KIR2DL3 recognitionis dictated by the presence of an Asn residue at position 80.Importantly the great majority of HLA-C alleles have either an Asn or aLys residue at position 80. One KIR with three Ig domains, KIR3DL1(p70), recognizes an epitope shared by HLA-Bw4 alleles. Finally, ahomodimer of molecules with three Ig domains KIR3DL2 (p140) recognizesHLA-A3 and -A11.

Although KIRs and other class-I inhibitory receptors (Moretta et al,1997; Valiante et al, 1997; Lanier, 1998; the disclosures of which areincorporated herein by reference) may be co-expressed by NK cells, inany given individual's NK repertoire, there are cells that express asingle KIR and thus, the corresponding NK cells are blocked only bycells expressing a specific class I allele group. Accordingly, asdescribed infra, when inhibitory receptors are targeted, the presentmethods will often involve the administration of compounds that targetmultiple inhibitory receptors, thereby ensuring a broad-based effectthat reaches a maximum range of NK cells.

In certain embodiments, the compound, preferably an antibody or afragment thereof, blocks an inhibitory receptor of an NK cell,neutralizing the inhibitory signal of at least one inhibitory receptorselected from the group consisting of KIR2DL2, KIR2DL3, KIR2DL1,KIR3DL1, KIR3DL2, NKG2A and NKG2C. More preferably, the compound,preferably an antibody or a fragment thereof, that blocks the inhibitoryreceptor of an NK cell, is a compound, preferably an antibody or afragment thereof that neutralizes the inhibitory signal of KIR2DL2,KIR2DL 3 and/or KIR2DL1.

The invention also contemplates the use of a combination of severalcompounds, preferably antibodies or a fragment thereof, that blockdifferent inhibitory receptors of NK cells. Preferably, compounds,preferably antibodies or a fragment thereof, that block inhibitoryreceptors of NK cells are specific of an inhibitory receptor selectedfrom KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, NKG2A and NKG2C andare able to inhibit the related KIR- or NKG2A/C-mediated inhibition ofNK cell cytotoxicity. For example, the compounds that block inhibitoryreceptors of NK cells can comprise an antibody having a specificity forKIR2DL1 and an other having a specificity for KIR2DL2 and/or KIR2DL3.More preferably, the combination of compounds that block inhibitoryreceptors of NK cells is able to inhibit the KIR2DL1-, KIR2DL2-, andKIR2DL3-mediated inhibition of NK cell cytotoxicity. In otherembodiments, a cocktail of one or more compounds targeting one or moreinhibitory receptors, as well as one or more compounds targeting one ormore activating receptors, will be administered.

For example, monoclonal antibodies specific for KIR2DL1 have been shownto block the KIR2DL1 Cw4 (or the like) alleles (Moretta et al., 1993;the disclosure of which is incorporated herein by reference). In another example, monoclonal antibodies against KIR2DL2/3 have also beendescribed that block the KIR2DL2/3 HLACw3 (or the like) alleles (Morettaet al., 1993). Anti NKG2A antibodies have been shown to block theinhibitory interaction between NKG2A and HLA-E.

Optionally, the antibody can be selected from the group consisting ofGL183 (KIR2DL2, L3, available from Immunotech, France and BecktonDickinson, USA); EB6 (KIR2DL1, available from Immunotech, France andBeckton Dickinson, USA); AZ138 (KIR3DL1, available from Moretta et al,Univ. Genova, Italy); Q66 (KIR3DL2, available from Immunotech, France);Z270 (NKG2A, available from Immunotech, France); P25 (NKG2A/C, availablefrom Moretta et al., Univ. Genova, Italy); and DX9, Z27 (KIR3DL1,available from Immunotech, France and Beckton Dickinson, USA).

In a preferred aspect, the invention uses monoclonal antibodies, as wellas fragments and derivatives thereof, wherein said antibody, fragment orderivative cross reacts with several KIR or NKG2A/C receptors at thesurface of NK cells and neutralizes their inhibitory signals.

In one embodiment, the invention uses a monoclonal antibody that binds acommon determinant of human KIR2DL receptors and inhibit thecorresponding inhibitory pathway. Preferably, the invention uses amonoclonal antibody that binds KIR2DL1 and KIR2DL2/3 receptors at thesurface of human NK cells and inhibits KIR2DL1- and KIR2DL2/3-mediatedinhibition of NK cell cytotoxicity. The antibody specifically inhibitsbinding of HLA-c molecules to KIR2DL1 and KIR2DL2/3 receptors. Morepreferably, the antibody facilitates NK cell activity in vivo. BecauseKIR2DL1 and KID2DL3 (or KIR2DL2) are sufficient for covering most of theHLA-C allotypes, respectively group 1 HLA-C allotypes and group 2 HLA-Callotypes, such antibodies may be used to increase the efficiency of atherapeutic antibody in most human individuals, typically in about 90%of human individuals or more. In such an embodiment, any of theantibodies described in PCT Patent Application no. PCT/FR 04/01702 filedJul. 1, 2004, titled “Compositions and methods for regulating NK cellactivity” can be used in accordance with the invention, the disclosureof which is incorporated herein by reference.

In a particular object of this invention, the antibody that blocks theinhibitory receptor of an NK cell is a monoclonal antibody, wherein saidantibody binds a common determinant of KIR2DL human receptors andinhibits KIR2DL-mediated inhibition of NK cell cytotoxicity. Theantibody more specifically binds to the same epitope as monoclonalantibody DF200 or NKVSF1 produced by hybridoma DF200 and NKVSF1respectively and/or competes with monoclonal antibody DF200 or NKVSF1produced by hybridoma DF200 and NKVSF1 respectively, for binding to aKIR receptor at the surface of a human NK cell. As discussed, examplesof antibodies, functional assays and assays to determine whetherantibodies compete for binding with said antibodies are described in PCTPatent Application no. PCT/FR 04/01702.

In a specific embodiment, the monoclonal antibody is monoclonal antibodyDF200 produced by hybridoma DF200. In another embodiment, the monoclonalantibody is EB6, or the antibody binds to the same epitope as monoclonalantibody EB6, or competes for binding with monoclonal antibody EB6. Inother embodiments, the antibody is a fragment or derivative of either ofantibodies DF200 or EB6. The hybridoma producing antibody DF200 has beendeposited at the CNCM culture collection, as Identification no. “DF200”,registration no. CNCM I-3224, registered 10 Jun. 2004, CollectionNationale de Cultures de Microorganismes, Institut Pasteur, 25, Rue duDocteur Roux, F-75724 Paris Cedex 15, France. The antibody NKVSF1 isavailable from Serotec (Cergy Sainte-Christophe, France), Catalog refno. MCA2243.

In another embodiment of the present invention, the compound used toenhance the efficacy of therapeutic antibodies stimulates an activatingreceptor of an NK cell. Any activating receptor can be used, e.g., NKp30(see, e.g., PCT WO 01/36630, the disclosure of which is hereinincorporated by reference in its entirety), NKp44 (see, e.g., Vitale etal. (1998) J. Exp. Med. 187:2065-2072, the disclosure of which is hereinincorporated by reference in its entirety), NKp46 (see, e.g., Sivori etal. (1997) J. Exp. Med. 186:1129-1136; Pessino et al. (1998) J. Exp.Med. 188:953-960; the disclosures of which are herein incorporated byreference in their entireties), NKG2D (see, e.g., OMIM 602893), IL-12R,IL-15R, IL-18R, IL-21R, or an activatory KIR receptor, for example aKIR2DS4 receptor (Carrington and Norman, The KIR Gene Cluster, May 3,2003, available at: worldwide web site ncbi.nlm.nih.gov/books), or anyother receptor present on a substantial fraction of NK cells, and whoseactivation leads to the activation or proliferation of the cell,preferably even if the cell had previously been inhibited via aninhibitory receptor such as an inhibitory KIR receptor. The compound canbe any molecular entity, including polypeptides, small molecules, andantibodies. Exemplary compounds include any ligands, including natural,recombinant or synthetic ligands, which interact with activatingreceptors. For example, a compound which stimulates an activatingreceptor of an NK cell may be a cytokine such as IL-12 which interactswith the IL-12 receptor (IL-12R), IL-15 which interacts with the IL-15receptor (IL-15R), IL-18 which interacts with the IL-18 receptor(IL-18R), IL-21 which interacts with the IL-21 receptor (IL-21R). Suchcompounds are known from e.g., IL-12 (Research Diagnostics, NJ, DI-212),IL-15 (Research Diagnostics, NJ, RDI-215), IL-21 (Asano et al, FEBSLett. 2002; 528:70-6). Preferably, a compound which stimulates anactivating receptor of an NK cell is a compound other than IL-2. Otherexemplary compounds which stimulate an activating receptor of an NK cellinclude antibodies which bind an NK cell receptor selected from thegroup consisting of NKp30, NKp44, NKp46, NKG2D, KIR2DS4 and otheractivatory KIR receptors.

In certain preferred embodiments, the activatory receptor is a NaturalCytotoxicity Receptor (NCR) found on NK cells, preferably the NCRselected from the group consisting of NKp30, NKp44 or NKp46, and thecompound that stimulates an activating receptor is, binds to the sameepitope as, or competes for binding with any of the monoclonalantibodies selected from the group consisting of AZ20, A76, Z25, Z231,and BAB281.

The binding of any compound to any of the herein-described NK cellreceptors can be detected using any of a variety of standard methods.For example, colorimetric ELISA-type assays can be used, as canimmunoprecipitation and radioimmunoassays. Competition assays may beemployed, e.g., to compare the binding of a test compound to a compoundknown to bind to an NK cell receptor, in which the control (e.g.,BAB281, which specifically binds to NKp46) and test compounds areadmixed (or pre-adsorbed) and applied to a sample containing theepitope-containing protein, e.g., NKp46 in the case of BAB₂₈₁. Protocolsbased upon ELISAs, radioimmunoassays, Western blotting and the use ofBIACORE are suitable for use in such simple competition studies and arewell known in the art.

Inhibition of KIR- or NKG2A/C-mediated inhibition of NK cellcytotoxicity, or stimulation of NKp30, NKp44, NKp46, or NKG2D-mediatedactivation of NK cells, can be assessed by various assays or tests, suchas binding, cytotoxicity, or other molecular or cellular assays.

In a specific variant, inhibitory activity is illustrated by thecapacity of said compound, preferably an antibody, to reconstitute thelysis of KIR or NKG2A/C positive NK clones, respectively, on HLA-C orHLA-E positive targets. In another specific embodiment, the compound,preferably an antibody, is defined as inhibiting the binding of HLA-Cmolecules to KIR2DL1 and KIR2DL3 (or the closely related KIR2DL2)receptors, further preferably as its capacity to alter the binding of aHLA-C molecule selected from Cw1, Cw3, Cw7, and Cw8 (or of a HLA-cmolecule having an Asn residue at position 80) to KIR2DL2/3; and thebinding of a HLA-C molecule selected from Cw2, Cw4, Cw5 and Cw6 (or of aHLA-c molecule having a Lys residue at position 80) to KIR2DL1.

The inhibitory or potentiating activity of a compound of this invention,preferably an antibody, can be assessed in any of a number of ways,e.g., by its effect on intracellular free calcium as described, e.g., inSivori et al. (1997) J. Exp. Med. 186:1129-1136, the disclosure of whichis herein incorporated by reference. NK cell activity can also beassessed using a cell based cytotoxicity assays, e.g., measuringchromium release, such as assessing the ability of the antibody tostimulate NK cells to kill target cells such as P815, K562 cells, orappropriate tumor cells as disclosed in Sivori et al. (1997) J. Exp.Med. 186: 1129-1136; Vitale et al. (1998) J. Exp. Med. 187: 2065-2072;Pessino et al. (1998) J. Exp. Med. 188: 953-960; Neri et al. (2001)Clin. Diag. Lab. Immun. 8:1131-1135); Pende et al. (1999) J. Exp. Med.190: 1505-1516), the entire disclosures of each of which are hereinincorporated by reference. Suitable cytotoxicity assays are alsoprovided in the examples section of the present specification. In apreferred embodiment, the antibodies cause at least a 10% augmentationin NK cytotoxicity, preferably at least a 40% or 50% augmentation in NKcytotoxicity, or more preferably at least a 70% augmentation in NKcytotoxicity.

NK cell activity can also be addressed using a cytokine-release assay,wherein NK cells are incubated with the antibody to stimulate the NKcells' cytokine production (for example IFN-γand TNF-α production). Inan exemplary protocol, IFN-γ production from PBMC is assessed by cellsurface and intracytoplasmic staining and analysis by flow cytometryafter 4 days in culture. Briefly, Brefeldin A (Sigma Aldrich) is addedat a final concentration of 5 μg/ml for the last 4 hours of culture. Thecells are then incubated with anti-CD3 and anti-CD56 mAb prior topermeabilization (IntraPrep™; Beckman Coulter) and staining withPE-anti-IFN-γ or PE-IgG1 (Pharmingen). GM-CSF and IFN-γ production frompolyclonal activated NK cells are measured in supernatants using ELISA(GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, Minn.; IFN-γ: OptE1Aset, Pharmingen).

In a preferred embodiment, the ability of the antibody to activate humanNK cells is assessed, where an ability to activate human NK cells atleast as well as non-human NK cells indicates that the compounds aresuitable for use in the present invention. In particular, the ability ofthe compound to enhance the ability of therapeutic antibodies to directthe depletion of suitable target cells by NK cells in vitro or in vivocan be assessed.

The compounds of this invention, preferably antibodies, may exhibitpartial inhibitory or stimulating activity, e.g., partially reduce theKIR2DL-mediated inhibition of NK cell cytotoxicity, or partiallyactivate an NK cell through any level of stimulation of NCRs or otherreceptors. Most preferred compounds are able to inhibit (or stimulate,in the case of activating receptors) at least 20%, preferably at least30%, 40% or 50% or more of the activity of the NK cell, e.g., asmeasured in a cell toxicity assay, in comparison to cells in the absenceof the compound. Also preferred, the compound can provide an increase ofdepletion of target cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 400%, 500%, 1000%, or more relative to thedepletion level in the absence of the compound. Alternatively, preferredcompounds of this invention, preferably antibodies, are able to inducethe lysis of matched or HLA compatible or autologous target cellpopulation, i.e., cell population that would not be effectively lysed byNK cells in the absence of said antibody. Accordingly, compounds of thisinvention may also be defined as facilitating NK cell activity in vivo.

The invention also contemplates embodiments in which compounds thatstimulate activating receptors, or, preferably, block the inhibitoryreceptor of an NK cell, are fragments of such a monoclonal antibodyhaving substantially the same antigen specificity, including, withoutlimitation, a Fab fragment, a Fab′2 fragment, a CDR and a ScFv.Furthermore, the monoclonal antibody may be humanized, human, orchimeric (e.g., a bispecific or functionalised antibody). Whileantibodies stimulating activating receptors can also be fragments, theyare preferably full length. Derivatives, e.g., with modified sequencesor with conjugated heterologous functional groups or other compounds,can be used for any of the antibodies described herein.

The antibodies that block the inhibitory receptor or stimulate anactivating receptor of an NK cell according to the invention may beproduced by a variety of techniques known in the art. Typically, theyare produced by immunization of a non-human animal with an immunogencomprising a KIR, NKG2A/C, NCR (e.g., NKp30, NKp44, NKp46), or NKG2Dpolypeptide, or immunogenic fragment of any of the polypeptides, andcollection of spleen cells (to produce hybridomas by fusion withappropriate cell lines). Methods of producing monoclonal antibodies fromvarious species are well known in the art (see, e.g., Harlow et al.,“Antibodies: A laboratory Manual,” CSH Press, 1988; Goding, “MonoclonalAntibodies: Principles and Practice,” Academic Press, 1986; thedisclosures of which are incorporated herein by reference). Morespecifically, these methods comprise immunizing a non-human animal withthe antigen, followed by a recovery of spleen cells which are then fusedwith immortalized cells, such as myeloma cells. The resulting hybridomasproduce the monoclonal antibodies and can be selected by limitingdilutions to isolate individual clones. Antibodies may also be producedby selection of combinatorial libraries of immunoglobulins, as disclosedfor instance in Ward et al. (1989); the disclosure of which isincorporated herein by reference.

Preferred antibodies that block the inhibitory receptor or stimulate anactivating receptor of an NK cell according to the invention areprepared by immunization with an immunogen comprising an activating orinhibiting NK cell receptor, e.g., a KIR2DL polypeptide, more preferablya human KIR2DL polypeptide. The KIR2DL polypeptide may comprise the fulllength sequence of a human KIR2DL polypeptide, or a fragment orderivative thereof, typically an immunogenic fragment, i.e., a portionof the polypeptide comprising an epitope, preferably a T or B cellepitope. Such fragments typically contain at least 7 consecutive aminoacids of the mature polypeptide sequence, even more preferably at least10 consecutive amino acids thereof. They are essentially derived fromthe extra-cellular domain of the receptor. In a preferred embodiment,the immunogen comprises a wild-type human KIR2DL, NCR, or otherpolypeptide in a lipid membrane, typically at the surface of a cell. Ina specific embodiment, the immunogen comprises intact NK cells,particularly intact human NK cells, optionally treated or lysed.

While the therapeutic antibodies may have Fe regions modified so as toenhance their binding by receptors such as CD16, in certain embodimentsNK cell potentiating antibodies will have Fc regions altered so as toreduce their affinity for Fc receptors, thereby reducing the likelihoodthat NK cells bound by the antibodies will themselves be bound andlysed.

Antibodies that block the KIR2DL receptors of NK cells can be producedby methods comprising: i) immunizing a non-human mammal with animmunogen comprising a KIR2DL polypeptide; ii) preparing monoclonalantibodies from said immunized animal, wherein said monoclonalantibodies bind said KIR2DL polypeptide; iii) selecting monoclonalantibodies from step ii) that cross react with at least two differentserotypes of KIR2DL polypeptides; and iv) selecting monoclonalantibodies of (c) that inhibit KIR2DL-mediated inhibition of NK cells.

The order of steps (iii) and (iv) can be changed. Optionally, the methodmay further comprise additional steps of making fragments or derivativesof the monoclonal antibody, as disclosed below.

In an other variant, the method comprises: i) selecting, from a libraryor repertoire, a monoclonal antibody or a fragment or derivative thereofthat cross reacts with at least two different serotypes of KIR2DLpolypeptides; and selecting an antibody from step i) that inhibitsKIR2DL-mediated inhibition of NK cells.

It will be appreciated that any of these methods can be used to selectfor any antibodies or anybody fragments that are specific for any groupof (inhibitory or activating) NK cell receptors sharing one or moreepitopes. For example, similar methods can be used for the preparationof antibodies that block a KIR3DL or a NKG2A/C receptor of NK cells, orstimulate an activating receptor of NK cells.

In preferred embodiment, the non-human animals used in these methods, orused in the production of any of the herein-described antibodies, is amammal, such as a rodent (e.g., mouse, rat, etc.), bovine, porcine,horse, rabbit, goat, sheep, etc.

Also, any of the herein-described antibodies can be genetically modifiedor engineered to be human-suitable, e.g., humanized, chimeric, or humanantibodies. Methods for humanizing antibodies are well known in the art.Generally, a humanized antibody according to the present invention hasone or more amino acid residues introduced into it from the originalantibody. These murine or other non-human amino acid residues are oftenreferred to as “import” residues, which are typically taken from an“import” variable domain. Humanization can be essentially performedfollowing the method of Winter and co-workers (Jones et al. (1986)Nature 321:522; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al.(1988) Science 239:1534 (1988)). In some cases, such “humanized”antibodies are chimeric antibodies (Cabilly et al., U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from theoriginal antibody. In practice, humanized antibodies according to thisinvention are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in the original antibody.

Another method of making “humanized” monoclonal antibodies is to use aXenoMouse® (Abgenix, Fremont, Calif.) as the mouse used forimmunization. A XenoMouse is a murine host that has had itsimmunoglobulin genes replaced by functional human immunoglobulin genes.Thus, antibodies produced by this mouse or in hybridomas made from the Bcells of this mouse, are already humanized. The XenoMouse is describedin U.S. Pat. No. 6,162,963, which is herein incorporated in its entiretyby reference. An analogous method can be achieved using a HuMAb-Mouse™(Medarex).

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et al., Nature 362 (1993) 255), or by selection of ant bodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

The antibodies of the present invention may also be derivatized to“chimeric” antibodies (immunoglobulins) in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in the original antibody, while the remainder of the chain(s)is identical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such antibodies, so long as theyexhibit the desired biological activity (Cabilly et al., supra; Morrisonet al. (1984) Proc. Natl. Acad. Sci. 81:6851).

It will also be appreciated that when the compound that blocks theinhibitory receptor of an NK cell or stimulates an activatory receptorof an NK cell is an antibody, such antibody may by polyclonal or,preferably, monoclonal. The antibody may be produced by a hybridoma orby a recombinant cell engineered to express the desired variable andconstant domains. The antibody may be a single chain antibody or otherantibody derivative retaining the antigen specificity and the lowerhinge region or a variant thereof. The antibody may be a polyfunctionalantibody, recombinant antibody, humanized antibody, or a fragment orderivative thereof. Said fragment or a derivative thereof is preferablyselected from a Fab fragment, a Fab′2 fragment, a CDR and a ScFv.Preferably a fragment is an antigen-binding fragment. An antibody thatcomprises an antibody fragment may also include but are not limited tobispecific antibodies. One example is a bispecific antibody comprisingan antigen binding region specific for an activatory receptor and anantigen binding region specific for a tumor antigen (see PCT Publicationno. WO 01/71005, the disclosures of which are incorporated herein byreference).

Composition and Administration

The invention concerns a composition comprising at least one compound,preferably an antibody or a fragment thereof, that blocks the inhibitoryreceptor or stimulates an activating receptor of an NK cell, and atherapeutic antibody, the use of said composition for increasing theefficiency of the therapeutic antibody, for increasing ADCC in a subjecttreated with a therapeutic antibody, or for treating a subject having adisease, more particularly a disease requiring the depletion of thetargeted cells, preferably diseased cells such as virally-infectedcells, tumor cells or other pathogenic cells. Preferably, the disease isselected from the group consisting of a cancer, an auto-immune disease,an inflammatory disease, a viral disease. The disease also concerns agraft rejection, more particularly allograft rejection, and graft versushost disease (GVHD).

More particularly, the treatment of the disease requires the depletionof the targeted cells, preferably the diseased cells such asvirally-infected cells, tumor cells or other pathogenic cells.Preferably, the disease is a cancer, infectious or immune disease. Morepreferably, the disease is selected from the group consisting of acancer, an auto-immune disease, an inflammatory disease, a viraldisease. The disease also concerns a graft rejection, more particularlyallograft rejection, and graft versus host disease (GVHD).

Said diseases include neoplastic proliferation of hematopoietic cells.Optionally, said diseases are selected from the group consisting oflymphoblastic leukemia, acute or chronic myelogenous leukemia, Hodgkin'slymphoma, Non-Hodgkin's lymphoma, myelodysplastic syndrome, multiplemyeloma, and chronic lymphocytic leukemia. Said diseases also includeENT cancers, colorectal cancers, breast cancer, epithelial cancer. Saiddiseases include CMV infection, and hepatitis B. Said diseases includeCrohn's disease, rheumatoid arthritis, asthma, psoriasis, multiplesclerosis or diabetes. In particular, any disease listed in the tableprovided supra can be treated.

Said therapeutic antibody can be bound by CD16, preferably through itsFc region. Preferably, said therapeutic antibody has a human IgG1 or anIgG3 Fc portion, particularly a monoclonal antibody or a fragmentthereof, further preferably a human, humanized or chimeric antibody or afragment thereof, for instance rituximab.

Said compound, preferably an antibody or a fragment thereof, that blocksthe inhibitory receptor or stimulates an activating receptor of an NKcell binds at least one of KIR, NKG2A/C, NCR, or NKG2D human receptors,and either inhibits the related KIR2DL, KIR3DL and/or NKG2A/C-mediatedinhibition of NK cell cytotoxicity, or stimulates the related NCR orNKG2D-mediated activation of NK cell cytotoxicity. In one preferredembodiment, a KIR2DL human receptor is used, e.g., a receptor selectedfrom the group consisting of KIR2DL1, KIR2DL2, KIR2DL3 human receptors,or a KIR3DL human receptor is used, e.g., a receptor selected from thegroup consisting of KIR3DL1 and KIR3DL2.

In one preferred embodiment, the NK-cell potentiating compound binds atleast one of KIR2DL human receptors and inhibits the relatedKIR2DL-mediated inhibition of NK cell cytotoxicity. Preferably, theKIR2DL human receptor is selected from the group consisting of KIR2DL1,KIR2DL2, KIR2DL3 human receptors. In a preferred embodiment, thecompound, preferably an antibody or a fragment thereof, binds a commondeterminant of KIR2DL human receptors and inhibits KIR2DL-mediatedinhibition of NK cell cytotoxicity. More preferably, said compound,preferably an antibody, binds a common determinant of KIR2DL1, KIR2DL2,KIR2DL3 human receptors and inhibits KIR2DL1-, KIR2DL2-,KIR2DL3-mediated inhibition of NK cell cytotoxicity. In a particularembodiment, said compound, preferably an antibody, inhibits the bindingof a HLA-C allele molecule having a Lys residue at position 80 to ahuman KIR2DL1 receptor, and the binding of a HLA-C allele moleculehaving an Asn residue at position 80 to human KIR2DL2 and KIR2DL3receptors. In another particular embodiment, this antibody binds to thesame epitope as monoclonal antibody DF200 produced by hybridoma DF200.Optionally, this antibody competes with monoclonal antibody DF200produced by hybridoma DF200 for binding to a KIR receptor at the surfaceof a human NK cell. In one preferred embodiment, the antibody ismonoclonal antibody DF200 produced by hybridoma DF200. In anotherembodiment, the antibody is, competes with, or binds to the same epitopeas monoclonal antibody EB6.

The composition according to the present invention can comprise acombination of several compounds, preferably antibodies or a fragmentthereof, that block different inhibitory receptors of NK cells, and/orstimulate one or more activating receptors of NK cells. Preferably,compounds, preferably antibodies or a fragment thereof, that blockinhibitory receptors of NK cells are specific of an inhibitory receptorselected from KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, NKG2A andNKG2C, and are able to inhibit the related KIR- or NKG2A/C-mediatedinhibition of NK cell cytotoxicity. More preferably, the combination of“neutralizing” compounds is able to inhibit the KIR2DL1-, KIR2DL2-, andKIR2DL3-mediated inhibition of NK cell cytotoxicity. By providing acombination of compounds, a maximum number of different inhibitoryreceptors will be blocked in a maximum number of patients. Also,combinations of compounds that stimulate different activating compounds(or, as with inhibitory receptors, bind to different epitopes within asingle receptor), can be used, e.g., compounds that together lead to theactivation of any combination of two or more receptors selected from thegroup consisting of NKp30, NKp44, NKp46, and NKG2D. Also, combinationscomprising one or more compounds that block an inhibitory receptor, andone or more compounds that stimulate an activating receptor, can beused. As described below, in a preferred embodiment, a sample of NKcells can be obtained from a patient prior to the application of thepresent methods, and the responsiveness of the NK cells to differentcombinations of compounds, e.g., in the presence of target cells and thetherapeutic antibody, can be assessed.

Compositions of this invention may comprise any pharmaceuticallyacceptable carrier or excipient, typically buffer, isotonic solutions,aqueous suspension, optionally supplemented with stabilizing agents,preservatives, etc. Typical formulations include a saline solution and,optionally, a protecting or stabilizing molecule, such as a highmolecular weight protein (e.g., human serum albumin).

Kits are also provided comprising any combination of one or moretherapeutic antibodies, one or more NK cell potentiating compounds, and,preferably, instructions for their use.

According to the methods and compositions of the present invention,compounds, preferably an antibody or a fragment thereof, that block aninhibitory receptor or stimulate an activating receptor of an NK celland therapeutic antibodies are administered in an “efficient” or“therapeutically effective” amount.

The efficient amount of therapeutic antibodies administered to therecipient can be between about 0.1 mg/kg and about 20 mg/kg. Theefficient amount of antibody depends however of the form of the antibody(whole Ig, or fragments), affinity of the mAb and pharmacokineticsparameter that must be determined for each particular antibodies.

The efficient amount of compounds, preferably an antibody or a fragmentthereof that block the inhibitory receptor or stimulate an activatingreceptor of an NK cell administered to the recipient can be betweenabout 0.1 mg/kg and about 20 mg/kg. The efficient amount of antibodydepends however of the form of the antibody (whole Ig, or fragments),affinity of the mAb and pharmacokinetics parameters that must bedetermined for each particular antibodies.

In an important embodiment of the invention, the use of the presentcompounds can allow therapeutic efficacy to be achieved with reduceddoses of therapeutic antibodies. The use (e.g., dosage, administrationregimen) of therapeutic antibodies can be limited by side effects, e.g.,in the case of rituximab, fever, headaches, wheezing, drop in bloodpressure, and others. Accordingly, while in many patients a standarddose of the therapeutic antibodies will be administered in conjunctionwith the herein-described NK cell potentiating compounds (i.e., therecommended dose in the absence of any other compounds), therebyenhancing the efficacy of the standard dose in patients needing evergreater therapeutic efficacy, in other patients, e.g., those severelyaffected by side effects, the administration of the present compoundswill allow therapeutic efficacy to be achieved at a reduced dose oftherapeutic antibodies, thereby avoiding side effects. In practice, askilled medical practitioner will be capable of determining the idealdose and administrative regimen of the therapeutic antibody and the NKcell potentiating compound for a given patient, e.g., the therapeuticstrategy that will be most appropriate in view of the particular needsand overall condition of the patient. Numerous references are availableto guide in the determination of proper dosages, for both thetherapeutic antibodies and the NK cell potentiating compounds, e.g.,Remington: The Science and Practice of Pharmacy, by Gennaro (2003),ISBN: 0781750253; Goodman and Gilmans The Pharmacological Basis ofTherapeutics, by Hardman, Limbird & Gilman (2001), ISBN: 0071354697;Rawlins E. A., editor, “Bentley's Textbook of Pharmaceutics”, London:Bailliere, Tindall and Cox, (1977); and others.

In one embodiment, a medical practitioner can gradually lower the amountof the therapeutic antibody given in conjunction with the administrationof any of the present NK cell potentiating compounds; either in terms ofdosage or frequency of administration, and monitor the efficacy of thetherapeutic antibody; e.g., monitor NK cell activity; monitor thepresence of target cells in the patient, monitor various clinicalindications, or by any other means, and, in view of the results of themonitoring, adjust the relative concentrations or modes ofadministration of the therapeutic antibodies and/or NK potentiatingcompound to optimize therapeutic efficacy and limitation of sideeffects.

In another set of embodiments, NK cells will be obtained from thepatient prior to the administration of the therapeutic antibody and NKcell potentiating compounds (and, if appropriate, during the treatment),and assessed to determine the ideal compound or suite of compounds to beused for maximum efficacy. For example, the identity of the inhibitoryor activating receptors present on the NK cells can be determined, andcompounds administered that specifically targeted the most prominentreceptors. Alternatively, the obtained NK cells can be incubated withthe therapeutic antibody and target cells, and the ability of one ormore compounds to enhance target cell depletion can be assessed.Whichever one or more compounds is most effective at enhancing depletionin vitro can then be selected for use in the present treatment methods.

Suitable doses of the compounds and/or therapeutic antibodies can alsogenerally be determined in vitro or in animal models, e.g., in vitro byincubating various concentrations of a therapeutic antibody in thepresence of target cells, NK cells (preferably human NK cells),optionally other immune cells, and varying concentrations of one or moreNK cell potentiating compounds, and assessing the extent or rate oftarget cell depletion under the various conditions, using standardassays (e.g., as described in the examples section). Alternatively,varying dosages of the therapeutic antibodies can be given to animalmodels for diseases treatable with the antibodies (e.g., an animal modelfor NHL in the case of rituximab), along with varying dosages of theherein-described compounds, and the efficacy of the antibodies (e.g., asdetermined by any suitable clinical, cellular, or molecular assay orcriterion) in treating the animals can be assessed.

The composition according to the present invention may be injecteddirectly to a subject, typically by intra-venous, intra-peritoneal,intra-arterial, intra-muscular or transdermic route. Several monoclonalantibodies have been shown to be efficient in clinical situations, suchas Rituxan (Rituximab) or Xolair (Omalizumab), and similaradministration regimens (i.e., formulations and/or doses and/oradministration protocols) may be used with the composition of thisinvention.

Furthermore, the compositions of this invention may further comprise ormay be used in combination with other active agents or therapeuticprograms such as chemotherapy or other immunotherapies, eithersimultaneously or sequentially.

In certain preferred example, the method of the invention furthercomprises one or several injections of two or more compounds that blockan inhibitory receptor or stimulate an activating receptor of an NKcell. Thus, these two or more compounds can be used in combination. Thiscan serve to cause an even greater augmentation of ADCC and efficacy oftherapeutic antibodies, and/or can serve to reduce the dosage of aparticular compound that block an inhibitory receptor or stimulate anactivating receptor of an NK cell. For example, compounds such as IL-2are known to be toxic at increased doses. The invention thereforepreferably provides a method of treatment of a disease in a subject inneed thereof comprising: a) administering to said subject at least twocompounds, preferably an antibody or a fragment thereof, that blocks aninhibitory receptor or stimulates an activating receptor of an NK cell;and b) administering to said subject a therapeutic antibody. Forexample, a preferred regimen is where said two compounds are (i) a firstcompound selected from the group consisting of an antibody whichstimulates an NCR or NKG2D receptor or an activatory KIR receptor, andan antibody which blocks an inhibitory KIR receptor or NKG2A, and (ii) asecond compound selected from the group consisting of IL-12, IL-15,IL-18 and IL-21. The invention therefore further provides a method oftreatment of a disease in a subject in need thereof comprising: a)administering to said subject a compound according to the invention,preferably an antibody or a fragment thereof, that blocks an inhibitoryreceptor or stimulates an activating receptor of an NK cell; and b)administering to said subject a therapeutic antibody; and (c)administering to said subject IL-2. IL2 is available from ResearchDiagnostics, NJ, RDI-202, or Chiron Corp. (Emeryville, Calif.).

The cytokine can be administered according to any suitableadministration regimen, and may be administered before, simultaneouslyand/or after administration of the compound which blocks an inhibitoryreceptor or stimulates an activating receptor of an NK cell, and before,simultaneously and/or after administration of therapeutic antibody. In atypical example, the cytokine is administered daily for a period of 5-10days, the cytokine(s) being first injected on the same day as the firstinjection of the compound which blocks an inhibitory receptor orstimulates an activating receptor of an NK cell. Said method preferablycomprises one or two injections/day of cytokine(s) by subcutaneousroute.

The dosage of the cytokine will be chosen depending on the condition ofthe patient to be treated. In preferred examples, a relatively low doseof cytokine can be used. For example, an effective dose of a cytokinesuch as IL-2 is typically lower than 1 million units/square meters/dayof cytokine(s), when the cytokine-containing pharmaceutical compositionis used for daily subcutaneous injection. In a preferred example, IL-2is injected subcutaneously at daily doses below 1 million units/m2 for 5to 10 days. Further detail of the use of cytokines is described inInternational Patent publication no. PCT/EP/0314716 and U.S. patentapplication No. 60/435,344 titled “Pharmaceutical compositions having aneffect on the proliferation of NK cells and a method using the same”,the disclosures of which are incorporated herein by reference. Cytokinescan be administered according to the manufacturer's instructions, andmodification to dosage and administration can be made as describedherein with respect to therapeutic antibodies.

It will also be appreciated that the therapeutic antibodies and NK cellpotentiating compounds can be coadministered, e.g., co-injected, or canbe administered simultaneously but in different formulations, or can beindependently administered, e.g., the compound is administered hours,days, or weeks before or after the administration of the compound.

Further aspects and advantages of this invention are disclosed in thefollowing experimental section, which should be regarded as illustrativeand not limiting the scope of this application.

EXAMPLES Example 1 Generation of a Pan KIR2DL Antibody

Purification of PBLs and Generation of Polyclonal or Clonal NK CellLines

PBLs were derived from healthy donors by Ficoll Hypaque gradients anddepletion of plastic adherent cells. To obtain enriched NK cells, PBLswere incubated with anti CD3, anti CD4 and anti HLA-DR mAbs (30 mns at4° C.), followed by goat anti mouse magnetic beads (Dynal) (30 mns at 4°C.) and immunomagnetic selection by methods known in the art (Pende etal., 1999). CD3 minus, CD4 minus DR minus cells are cultivated onirradiated feeder cells and 100 U/ml Interleukin 2 (Proleukin, ChironCorporation) and 1.5 ng/ml Phytohemagglutinin A (Gibco BRL) to obtainpolyclonal NK cell populations. NK cell are cloned by limiting dilutionand clones of NK cells are characterized by flow cytometry forexpression of cell surface receptors.

The following clones were used in this study:

CP11, CN5 and CN505 are KIR2DL1 positive clones and are stained by EB6or XA-141 antibodies. CN12 and CP502 are KIR2DL3 positive clones and arestained by GL183 antibody.

Flow Cytometry Analysis

Monoclonal antibodies (mAbs) used were produced in the laboratory JT3A(IgG2a, anti CD3), EB6 and GL183 (IgG1 anti KIR2DL1 and KIR2DL3respectively), XA-141 IgM anti KIR2DL1 (same specificity as compared toEB6, anti CD4 (HP2.6), anti DR (D1.12, IgG2a). Instead of JT3A, HP2.6,and DR1.12, commercially available mAbs of the same specificities can beused for example from Beckman coulter Inc, Fullerton, Calif. EB6 andGL183 are commercially available in Beckman Coulter Inc, Fullerton,Calif. XA-141 is not commercially available but EB6 can be used forcontrol reconstitution of lysis as described in (Moretta et al., 1993).

Flow Cytometry

Cells were stained with the appropriate antibodies (30 mns at 4° C.)followed by PE or FITC conjugated polyclonal anti mouse antibodies(Southern Biotechnology Associates Inc). Samples were analysed bycytofluorometric analysis on a FACSAN apparatus (Becton Dickinson,Mountain View, Calif.).

Cytotoxicity Experiments

The cytolytic activity of NK clones was assessed by a standard 4 hr 51Crrelease assay. In which effector NK cells were tested on Cw3 or Cw4positive cell lines known for their sensitivity to NK cell lysis. Allthe targets are used at 5000 cells per well in microtitration plate andthe Effector on target ratio is indicated in the figures (usually 4effectors per target cells). The cytolytic assay is performed with orwithout supernatant of indicated monoclonal antibodies at a 1/2dilution. The procedure is essentially the same as described in (Morettaet al., 1993).

Generation of New mAbs

Monoclonal antibodies (mAbs) have been generated by immunizing5-week-old Balb C mice with activated polyclonal or monoclonal NK celllines as described in (Moretta et al., 1990; the disclosure of which isincorporated herein by reference). After different cell fusions, themAbs were first selected for their ability to cross react with EB6 andGL183 positive NK cell lines and clones. Positive monoclonal antibodieswere further screened for their ability to reconstitute lysis by EB6positive or GL183 positive NK clones of Cw4 or Cw3 positive targetsrespectively.

DF200, a novel monoclonal antibody against a common determinant ofKIR2DL human NK receptors

One of the monoclonal antibodies, the DF200 mAb, was found to react withvarious members of the KIR family, including: KIR2DL1 and KIR2DL2/3.Regarding the staining of NK cells with DF200 mAb, both KIR2DL1+ andKIR2DL2/3+ cells were stained brightly (FIG. 1).

NK clones expressing one or another (or even both) of these HLA classI-specific inhibitory receptors were used as effectors cells againsttarget cells expressing one or more HLA-C alleles. As expected,KIR2DL1+NK clones displayed little if any cytolytic activity againsttarget cells expressing HLA-Cw4 and KIR2DL3+NK clones displayed littleor no activity on Cw3 positive targets. However, in the presence ofDF200 mAb (used to mask their KIR2DL receptors) NK clones became unableto recognize their HLA-C ligands and displayed strong cytolytic activityon Cw3 or Cw4 targets.

For example, the C1R cell line (CW4+EBV cell line, ATCC n° CRL 1993) wasnot killed by KIR2DL1+NK clones (CN5/CN505), but the inhibition could beefficiently reverted by the use of either DF200 or a conventional antiKIR2DL1 mAb. On the other hand NK clones expressing theKIR2DL2/3+KIR2DL1-phenotype (CN12) efficiently killed C1R and thiskilling was unaffected by the DF200 mAb (FIG. 2). Similar results can beobtained with KIR2DL2- or KIR2DL3-positive NK clones on Cw3 positivetargets.

Biacore analysis of DF200 mAb/KIR 2DL1 and DF200 mAb/KIR 2DL3interactions

Materials and Methods

Production and purification of recombinant proteins. The KIR 2DL1 andKIR 2DL3 recombinant proteins were produced in E. coli. cDNA encodingthe entire extracellular domain of KIR 2DL1 and KIR 2DL3 were amplifiedby PCR from pCDM8 clone 47.11 vector (Biassoni et al, 1993; thedisclosure of which is incorporated herein by reference) andRSVS(gpt)183 clone 6 vector (Wagtman et al, 1995; the disclosure ofwhich is incorporated herein by reference) respectively, using thefollowing primers:

Sense: (SEQ ID NO: 1) 5′-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3′Anti-sense: (SEQ ID NO: 2) 5′-CCCAAGCTTGGGTTATGTGACAGAAACAAGCAGTGG-3′

They were cloned into the pML 1 expression vector in frame with asequence encoding a biotinylation signal (Saulquin et al, 2003; thedisclosure of which is incorporated herein by reference).

Protein expression was performed into the BL21(DE3) bacterial strain(Invitrogen). Transfected bacteria were grown to OD₆₀₀=0.6 at 37° C. inmedium supplemented with ampicillin (100 μg/ml) and induced with 1 mMIPTG.

Proteins were recovered from inclusion bodies under denaturingconditions (8 M urea). Refolding of the recombinant proteins wasperformed in Tris 20 mM, pH 7.8, NaCl 150 mM buffer containingL-arginine (400 mM, Sigma) and β-mercaptoethanol (1 mM), at RT, bydecreasing the urea concentration in a six step dialysis (4, 3, 2, 1 0.5and 0 M urea, respectively). Reduced and oxidized glutathion (5 mM and0.5 mM respectively, Sigma) were added during the 0.5 and 0 M ureadialysis steps. Finally, the proteins were dialyzed extensively againstTris 10 mM, pH 7.5, NaCl 150 mM buffer. Soluble refolded proteins wereconcentrated and then purified on a Superdex 200 size-exclusion column(Pharmacia; AKTA system).

Biacore analysis. Surface plasmon resonance measurements were performedon a Biacore apparatus (Biacore). In all Biacore experiments HBS buffersupplemented with 0.05% surfactant P20 served as running buffer.

Protein immobilization. Recombinant KIR 2DL1 and KIR 2DL3 proteins wereimmobilized covalently to carboxyl groups in the dextran layer on aSensor Chip CM5 (Biacore). The sensor chip surface was activated withEDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride andN-hydroxysuccinimide, Biacore). Proteins, in coupling buffer (10 mMacetate pH 4.5) were injected. Deactivation of the remaining activatedgroups was performed using 100 mM ethanolamine pH8 (Biacore).

Affinity measurements. For kinetic measurements, various concentrationsof the soluble antibody (10⁻⁷ to 4×10⁻¹° M) were applied onto theimmobilized sample. Measurements were performed at 20 μl/min continuousflow rate. For each cycle, the surface of the sensor chip wasregenerated by 5 μl injection of 10 mM NaOH pH 11.

The BIAlogue Kinetics Evaluation program (BIAevaluation 3.1, Biacore)was used for data analysis.

Results

BIAcore analysis of DF200 mAb binding to immobilized KIR 2DL1 and KIR2DL3.

KD (10⁻⁹M) KIR 2DL1 10.9 +/− 3.8 KIR 2DL3  2.0 +/− 1.9 KD: Dissociationconstant.

The soluble analyte (40 ml at various concentrations) was injected at aflow rate of 20 μl/min in HBS buffer, on a dextran layers containing 500or 540 reflectance units (RU), and 1000 or 700 RU of KIR 2DL1 and KIR2DL3 respectively. Data are representative of 6 independent experiments.

Example 2 Enhancement of ADCC by Using a Combination of Rituxan and AntiKIR mAb

Preparation of human NK clones. Blood mononuclear cells depleted of Tcells by negative anti-CD3 immuno-magnetic selection (Miltenyi) areplated under limiting-dilution conditions, activated withphytohemagglutinin (PHA) (Biochrom KG, Berlin, Germany), and culturedwith interleukin (IL)-2 (Chiron B.V., Amsterdam, Netherlands) andirradiated feeder cells. Cloning efficiencies are equivalent in alldonors and range between 1 in 5 and 1 in 10 plated NK cells. Cloned NKcells are screened for alloreactivity by standard 51Cr releasecytotoxicity against Epstein-Barr virus-transformed B lymphoblastoidcell lines of known HLA type at an effector to target ratio of 10:1.Clones exhibiting≧30% lysis were scored as alloreactive. As a rule,clones either exhibit <5% or >40% lysis.

Enhancement of ADCC mediated by Rituxan by a KIR2DL1 positive NK cellclone

The cytolytic activity of NK clone is assessed by a standard 4 hr 51Crrelease assay, in which effector NK cells were tested on Cw4 or Cw3positive EBV cell lines (CD20 positive), known for their sensitivity toNK cell lysis. All the targets are used at 5000 cells per well inmicrotitration plate and the Effector (NK cell clone) on target ratio isindicated in FIG. 3. In certain experiments, the therapeutic chimericanti CD20 rituximab (Rituxan, Idec) is added at 5 μg/ml is added to theeffector target mixture. In certain experiments, the EB6 antibody (antiKIR2DL1) at 10 μg/ml is added to the effector target mixture.

This experiment showed that Rituxan alone mediates essentially no ADCCby the KIR2DL1 positive NK clone on Cw4 positive target. ADCC of KIR2DL1positive clone is greatly enhanced in the presence of anti KIR2DL1antibody.

Example 3 Enhancement of ADCC Mediated by Campath by a Kir2DL1 PositiveNk Cell Clone

In a similar experiment to that described in Example 2, autologousCw4+PHA blasts were incubated in the presence of NK cells plusalumtuzumab (Campath, Berlex), the EB6 antibody (at 100 ug/ml), orCampath and EB6. The results, shown in FIG. 4, show that the presence ofEB6 dramatically enhances the ability of the NK cells to deplete theautologous cells: approximately 4% of the target cells were lysed in thepresence of Campath alone, whereas more than 30% of the cells were lysedin the presence of Campath plus EB6.

REFERENCES

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All publications and patent applications cited in this specification areherein incorporated by reference in their entireties as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext). This invention includes all modifications and equivalents ofthe subject matter recited in the aspects or claims presented herein tothe maximum extent permitted by applicable law.

1-25. (canceled)
 26. A method of treating a disease in a human subject comprising administering to a human subject in need thereof: (a) a therapeutically effective amount of a therapeutic antibody or antigen-binding fragment that specifically binds to an antigen that is expressed on target cells but not on NK cells, wherein the therapeutic antibody or antigen-binding fragment binds via its Fc region to CD16 and is capable of mediating depletion of the target cells by antibody-dependent cell-mediated cytotoxicity (ADCC); and (b) administering to a human subject in need thereof at least one NK cell-potentiating compound that specifically binds to and blocks an inhibitory receptor expressed on the surface of an NK cell, wherein the at least one NK cell-potentiating compound is present in an amount sufficient to enhance the efficacy of the therapeutic antibody or antigen-binding fragment by enhancing the depletion of the target cells by ADCC.
 27. The method of claim 26, wherein the therapeutic antibody or antigen-binding fragment comprises a human or non-human primate IgG1 or IgG3 Fe portion.
 28. The method of claim 26, wherein the at least one NK cell-potentiating compound comprises (i) an antibody or an antigen binding fragment thereof; (ii) a monoclonal antibody or comprises an antigen-binding fragment thereof; or (iii) a human, humanized or chimeric antibody or comprises an antigen-binding fragment thereof.
 29. The method of claim 26, wherein the therapeutic antibody is a monoclonal antibody or comprises an antigen-binding fragment thereof.
 30. The composition of claim 29, wherein the monoclonal antibody is a human, humanized or chimeric antibody or comprises an antigen-binding fragment thereof.
 31. The method of claim 29 wherein the therapeutic antibody or antigen-binding fragment is not conjugated with a radioactive or toxic moiety.
 32. The method of claim 26, wherein the therapeutic antibody is rituximab or alemtuzumab.
 33. The composition of claim 28, wherein the at least one NK cell-potentiating compound is (i) an antibody or antigen binding fragment thereof that binds to and blocks an inhibitory receptor expressed on the surface of an NK cell, wherein the inhibitory receptor is selected from the group consisting of KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, and LILRB5; or (ii) an antibody or antigen-binding fragment thereof that binds to a common determinant of KIR2DL human receptors and inhibits KIR2DL-mediated inhibition of NK cell cytotoxicity.
 34. The composition of claim 33, wherein the inhibitory receptor is KIR2DL1 or KIR2DL2/3.
 35. The method of claim 26, wherein the at least one NK cell-potentiating compound (i) binds to a common determinant of KIR2DL human receptors and inhibits KIR2DL-mediated inhibition of NK cell cytotoxicity; (ii) binds to a common determinant of KIR2DL1, KIR2DL2, and KIR2DL3 human receptors and inhibits KIR2DL1-, KIR2DL2-, and KIR2DL3-mediated inhibition of NK cell cytotoxicity; or (iii) inhibits the binding of an HLA-C polypeptide having a Lys residue at position 80 to a human KIR2DL1 receptor, and the binding of an HLA-C polypeptide having an Asn residue at position 80 to human KIR2DL2 and KIR2DL3 receptors.
 36. The method of claim 26, wherein the at least one NK cell-potentiating compound (i) binds to the same epitope as does monoclonal antibody DF200 produced by hybridoma DF200 (deposited as CNCM 1-3224), or as does monoclonal antibody EB6; (ii) competes with monoclonal antibody DF200 produced by hybridoma DF200 (deposited as CNCM 1-3224), or monoclonal antibody EB6, for binding to a KIR receptor expressed on the surface of a human NK cell; or (iii) comprises monoclonal antibody DF200 produced by hybridoma DF200 (deposited as CNCM 1-3224) or a fragment or derivative thereof, or monoclonal antibody EB6 or a fragment or derivative thereof.
 37. The method of claim 26, wherein the therapeutic antibody or antigen-binding fragment specifically binds to an antigen expressed on cells selected from tumor cells, virus-infected cells, allogeneic cells, non-tumorigenic pathological cells, pathological immunocompetent cells, and endothelial cells.
 38. The method of claim 26, wherein the at least one NK cell-potentiating compound enhances the ability of the therapeutic antibody to deplete said target cells by at least 30% or by at least 50%.
 39. The method of claim 26, wherein the therapeutic antibody or antigen-binding fragment elicits essentially no ADCC in the absence of the at least one NK cell-potentiating compound.
 40. The method of claim 26, wherein the therapeutic antibody or antigen-binding fragment thereof binds to an antigen selected from CD20, CD52, CD33, HLA-DR, CD22, HER2, erbB2, CA125, MUC1, PEM antigen, CD44, gp72, EpCAM, VEGFR, CD18, nuC242 EGFR, HER-1, CEA, αVfβ3, KDR (VEGFR2), VRS, CMV, HBs, CD25, TNF-α, CD80, IgE, CD11a(LFA-1), CD4, CD3, CD64, CD147, α4 β1-α4 β7, integrin β7, α4 β7, HLA-DR10 β, GD2, SK-1, IL-8, VLA-4, CD40L, E-selectin, CD11/CD18, ICAM-3, CBL, CD147, CD23, T1-ACY, TTs, CA19.9, PSA, HMFG1, hCH, collagen, CD46, 17A-1, HM1.24, CD38, IL-15R, Il-6, TRAIL-R1, VEGF2, B1yS, SCLS, Lewis Y antigen, VE cadherin, CD56, mertansine/mucine, AFP, CSap, CD30, PSMA, Cd15, CD19/CD3, mesothelin, DNA, histone, a5B1 integrin, p97, and CD5.
 41. The method of claim 26, wherein the target cells are tumor cells or virus-infected cells.
 42. The method of claim 37, wherein the pathological immunocompetent cells are B lymphocytes, T lymphocytes, or antigen-presenting cells.
 43. The method of claim 37, wherein the healthy cells are endothelial cells to be targeted in an anti-angiogenic therapeutic strategy.
 44. The method of claim 26, wherein the inhibitory receptor expressed on the surface of an NK cell is KIR2DL1 or KIR2DL2/3.
 45. The method of claim 26, wherein the inhibitory receptor expressed on the surface of an NK cell is selected from the group consisting of KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, and LILRB5.
 46. The method of claim 26, wherein said NK cell-potentiating compound enhances the ability of said therapeutic antibody to deplete said target cells by ADCC by at least 100%, by at least 200%, or by at least 300%.
 47. A method of selecting a compound for administration to a subject in conjunction with a therapeutic antibody or an antigen-binding fragment thereof, said method comprising: (i) providing a test compound that specifically binds to and blocks an inhibitory receptor expressed on the surface of an NK cell or specifically binds to and stimulates an activating receptor expressed on the surface of an NK cell; (ii) incubating a therapeutic antibody or antigen-binding fragment with target cells specifically recognized by the therapeutic antibody or antigen-binding fragment in the presence of NK cells and in the presence or absence of the test compound; and (iii) assessing the effect of the test compound on the ability of the NK cells to deplete target cells, wherein a detection that said compound enhances the ability of the NK cells to deplete target cells indicates that the test compound is suitable for administration to a subject in conjunction with the therapeutic antibody or antigen-binding fragment.
 48. A method of treating a disease in a human subject comprising administering to a human subject in need thereof: (a) a therapeutically effective amount of a therapeutic antibody or an antigen-binding fragment that specifically binds to an antigen that is expressed on target cells but not on NK cells, wherein the therapeutic antibody or antigen-binding fragment binds via its Fc region to CD16 and is capable of mediating depletion of the target cells by antibody-dependent cell-mediated cytotoxicity (ADCC); and (b) at least one NK cell-potentiating compound that specifically binds to and stimulates an activating receptor expressed on the surface of an NK cell, wherein the at least one NK cell-potentiating compound is present in an amount sufficient to enhance the efficacy of the therapeutic antibody or antigen-binding fragment by enhancing the depletion of the target cells by ADCC.
 49. The method of claim 48, wherein the activating receptor expressed on the surface of an NK cell is selected from the group consisting of NKp30, NKp44, NKp46, NKG2C, NKG2E, NKG2D, and KIR2DS4. 